The oxygen affinity of hemoglobin (Hb) is significantly influenced by several allosteric effectors including 2,3-diphosphoglycerate (2,3-DPG), protons, chloride, and carbon dioxide. A number of studies have characterized how these allosteric effectors modulate the reactivity of hemoglobin. The carbamino hemoglobin (HbNHCOO-) has been a difficult molecule to isolate; thus, the structure/function relationship of the CO2 binding to hemoglobin is not as well understood. One of the reasons for this is the lack of an optical signal with which to follow the reaction of carbon dioxide with hemoglobin. This is generally true with all of the effectors such as chloride, protons, etc. While the oxygen equilibrium curve can be measured as a function of oxygen and an effector, the reaction of the effector directly with hemoglobin can not. It is for this reason that several instruments which measure the reaction of the effectors by thermal means as well as optical or pH have been developed. The major task then of this program is to develop a set of instruments which can be readily operated by skilled technicians etc. so that hemoglobins, normal or modified, for use as a blood substitute can be readily characterized and their performance under both laboratory and in vivo conditions predicted. This year has mainly been taken up in getting the instruments set up, calibrated and running on a routine basis. Thus far an oxygen equilibrium apparatus for whole blood has been brought up and is currently wider test with whole blood. A second instrument for hemoglobin solution is under test. This is a more complex instrument and measures the curve by both manometric and spectroscopic means to try and settle several scientific issues. A very sensitive titrating flow calorimeter is presently being installed and tested and will be used for binding studies of effectors and toxins. A pulsed-flow differential optical-thermal flow apparatus is presently undergoing final tests and will be used for fast kinetic measurements of hemoglobin -carbon monoxide and oxygen studies with and without effectors. We feel that as the instruments become routinely operational they will provide us with all of the need characterizations to compare various hemoglobins, mutants, and modifieds.